Methods for the rapid and sensitive detection of nucleic acid fragments are gaining importance in the medical, environmental and food diagnostic industries as well as areas relying on genetic analysis such as forensics. Because of their specificity, nucleic acid sequences are being relied upon for the positive identification of disease as well as for indicating the presence of contaminating bacteria and other microorganisms.
Typically the identification of nucleic acid fragments has involved time consuming methods such as restriction enzyme analysis followed by gel electro-phoresis and staining. Such methods lack sensitivity and are not easily adaptable for rapid detection and field use.
With the development of techniques for rapid nucleic acid amplification involving thermostable nucleic acid amplifying enzymes (ligases, polymerases, transferases, etc.) such as the polymerase chain reaction (PCR), ligase chain reaction (LCR) and the relatively new strand displacement amplification (SDA), it is now possible to generate multiple copies of nucleic acid analytes that have been heretofore undetectable. Modifications of these techniques have allowed for the generation of ligand-labeled nucleic acid fragments which has in turn permitted the development of new, highly sensitive nucleic acid detection assays.
Recently developed assays use labeled nucleic acid fragments immobilized on various supports for detection of the desired fragment by an enzyme or fluorescent reporter. EP 437774 discloses single or double-stranded nucleic acids containing one or more detectable groups and two or more immobilization-facilitating groups in one strand. A target nucleic acid is amplified by a polymerase chain reaction forming multiple copies of a complementary nucleic acid containing detection or immobilizing groups. The resulting nucleic acid is immobilized on a solid phase and the detectable groups are detected. The immobilization-facilitating groups are derived from haptens such as digoxigenin and vitamins such as biotin, as well as antigens, antibodies, and lectins. The detectable groups are selected from radio-isotopes, fluorescent dyes, chromophores or indirectly-detectable groups such as digoxigenin and enzyme reporters.
EP 420260 teaches a hybridization method for the assay of nucleic acids involving a capture probe immobilized on a solid support to bind a labelled target nucleic acid sequence. A labelled target nucleic acid is amplified from a biological sample where the target is hybridized with at least one oligonucleotide capture probe having a nucleic acid sequence complementary to the target sequence, and where the capture probe is bound to a polystyrene solid support. Hybridization takes place in the presence of guanidine thiocyanate and the label may be biotin in the form of biotin-11-dUTP incorporated by Taq polymerase during polymerase chain reaction (PCR) amplification. The biotin label is detected by the addition of avidin or streptavidin complexed with horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, luciferase, fluorescein or Texas red. The HRP is detected with a chromogenic agent and H.sub.2 O.sub.2.
In a similar method, EP 447464 discloses a method for the capture and detection of ligand labeled DNA. The method involves capturing ligand containing amplified target DNA on a solid substrate having an immobilized binding reagent for the ligand. The binding reagent is a DNA binding protein, such as glutathione-5-transterase (GST)-GCN4 or Tyr R and detection of the captured DNA is accomplished with a second ligand such as biotin where detection is accomplished via an avidin/peroxidase system.
Hornes et al., in WO 9217609 teach a method of detecting bacterial cells using PCR of bacterial nucleic acids using ligand labeled nested primers. The method comprises lysing a mixture of bacterial cells and liberating DNA and/or RNA followed by amplifying DNA characteristic of the cells by PCR and detecting the amplified DNA. The PCR is carried out using nested primers, where one of the inner nested primers carries a biotin molecule and the other inner primer carries digoxigen.
The above cited methods are useful for detection of amplified nucleic acid analytes. However, they have several limitations including the need to perform the reactions by the sequential addition of reagents followed by multiple washings and the inability to assay for multiple analytes.
Methods of nucleic acid detection involving fewer washing steps and immobilization on a porous membrane have also been disclosed. Corti et al. (Nuc. Acids Res., 19, 1351 (1991) teach a method for the detection of DNA that is based on hybridization of target DNA with digoxigenin-lableled probes on disposable filters. The method involves prewetting the filter with buffer followed by addition of target DNA, addition of blocking reagent and hybridizing solution and finally the subsequent addition of digoxigenin-labelled DNA probe under hybridizing conditions. Detection of bound digoxigenin-DNA is carried out with alkaline phosphatase/anti-digoxigenin antibody conjugate and chomogenic substrates.
The method of Corti et al. is useful for detection of labeled DNA fragments, but is still hampered by the time consuming, sequential addition of reagents. A preferred method would derive benefit from a single addition of reagents and the elimination of washing steps.
The relative speed and specificity of immunoassays have lead to the development of test kits capable of rapid immunological diagnostic tests. Several of these immunoassays employ a lateral fluid flow system comprising a porous membrane material within which test reagents are located that are capable of reacting with analytes in a test solution as they are drawn down the membrane by capillary action.
Weng et al. (U.S. Pat. No. 4,740,468) disclose a method for determining the presence of an analyte in a sample involving contacting one end of a bibulous test strip with a test solution which contains the sample and a first member of a specific binding pair. The bibulous test strip is capable of being traversed by the test solution through capillary action. The first member of a specific binding pair is designed to specifically bind the analyte. The strip further contains a second member of a specific binding pair useful for concentrating and binding the first specific binding pair member at a small site on the strip. The detectable signal is produced in relation to the presence of the analyte in the test solution. The method of Weng et al. does not disclose or teach the detection of nucleic acids.
Similarly, May et al. (WO 8808534) disclose an analytical test device comprising a porous carrier material which contains a labelled binding reagent, specific for an analyte. This reagent is able to move freely within the carrier material. The strip also contains an unlabeled specific binding reagent for the same reagent which is immobilized in a detection zone on the carrier material. Liquid sample applied to the device is able to pick up labelled reagent and permeate the detection zone where it reacts with the unlabeled reagent and is immobilized for detection. A similar method is disclosed by Wood et al. (EP 170746) where an analyte is bound to a multiplicity of particles which are in turn adhered to a solid plastic support for detection and by Adams et al. (U.S. Pat. No. 4,189,304) who teach a method of detecting myoglobin in the presence of hemoglobin comprising a chromatographic medium and device.
The above cited immunological methods are useful for the detection and quantitation of immuno-reactive analytes. However, they do not teach the detection and identification of nucleic acid fragments.
In developing assays involving immunological reactions many factors must be considered. One consideration is to provide substantial differentiation between the observed signal resulting from signal label when bound as compared to unbound. Another consideration is to minimize interference from endogenous materials in the sample suspected of containing the compound of interest. Further considerations are the ease with which the observed signal can be detected and its ability to differentiate between concentrations in the concentration range of interest. Other factors include the ease of preparation of the reagents, the accuracy with which samples and reagent solutions must be prepared and measured, the storage stability of the reagents, the number of steps required in the protocol, and the proficiency and accuracy with which each of the steps must be performed. Therefore, in developing an assay for use by untrained personnel (such as assays to be performed in the home, in forensic medicine, by medical practitioners, or the like), the observed result should be minimally affected by variations in the manner in which the protocol is carried out or provide for simple techniques for performing the various steps.
There exists a need, therefore, for a method for the rapid, sensitive and facile identification of nucleic acid fragments that is readily adaptable for use in the field.